Over the past several years, we have been studying two phenomena in cloned populations of CD4+ T lymphocytes referred to as co-stimulation and anergy. Both affect the production of the T cell growth factor interleukin-2 (IL-2) produced by these cells. Co-stimulation entails a 30 to 100-fold enhancement of IL-2 production when signaling through the antigen-specific T cell receptor is supplemented with signaling through the CD28 receptor on the same cell. Anergy is an anti-proliferative state that the T cell enters when it only receives a signal through the antigen-specific receptor. In this case, subsequent stimulation of IL-2 production is inhibited 20-50-fold. Our goals are to try and understand the molecular mechanisms behind these two phenomena and to explore their relevance in vivo. During the past year, we have made progress in two areas. One is a characterization of the IL-2 enhancer response elements that are involved in anergy. Reporter constructs were used previously to identify a region around position -180 from the transcription start site as a critical control region for anergy, but our first attempts to demonstrate the binding of regulatory proteins to that region were unsuccessful. Now we have determined that the nature of the nonspecific competitor in the assay is critical and that if we convert from poly-dI-dC to poly-dG-poly-dC, we can detect in a gel mobility shift assay a protein complex binding to a double stranded oligonucleotide corresponding in sequence to the nucleotides in this region. Mutation of the residues around -180 eliminates this binding. Also, competition for binding of the labeled oligonucleotide with unlabelled oligonucleotides showed specific inhibition with oligos corresponding to the -180 site but not with oligos corresponding to other sites at -150 and -105 that have also been implicated in anergy. Our current studies are focused on identifying the nature of the proteins that bind to the -180 site. The second project is the characterization of an in vivo model for anergy. Injection of a T cell receptor (TCR) transgenic mouse three times at 5 day intervals with the superantigen staphylococcal enterotoxin A (SEA) induces a profound inhibition of IL-2 production when spleen or lymph node T cells are restimulated in vitro with either SEA or the specific antigen recognized by the transgenic TCR. Surprisingly, these T cells are capable of making IL-2 if stimulated under limiting dilution conditions where there is only one responding T cell per well. Increases in the cell number per well, however, reduced the frequency of IL-2 producing wells suggesting that a second cell exists in the population which suppresses IL-2 production This conclusion is supported by mixing experiments in which spleen cells from SEA-treated mice suppressed the antigen-induced proliferation and IL-2 production of cells from control TCR transgenic mice. Current studies are focused on characterizing the nature of the suppresser cell and the molecular basis of the suppression.